MECHANISMS OF CYTOSOLIC CHAPERONIN ACTION

描述：双环伴侣蛋白复合物是一种重要的介质， 细胞蛋白质折叠 基于它们能够将未折叠的 在它们的环腔中的多肽，它们阻止了非途径反应 并促进生产性蛋白质折叠到天然状态， ATP依赖性 伴侣蛋白腔出现部分折叠 然后将底物蛋白释放到溶液中。 功能 推导了细菌GroEL/GroES伴侣蛋白系统的原理 根据体外重建研究，其作用的确切机制是 但并不清楚。 与同源寡聚体GroEL不同， 发现的真核细胞溶质伴侣蛋白TRiC是一种异源寡聚体， 复杂，似乎不依赖于GroES类辅因子。 知之甚少 关于这种伴侣蛋白的功能和底物特异性。 目标 这一建议的关键是要了解分子机制， 伴侣蛋白环复合物介导胞质蛋白质折叠。 以下 将在体外用纯化的组分阐述具体方面： 固定化伴侣蛋白复合物和陷阱伴侣蛋白的折叠测定 将用于实验中，以确定GroEL/GroES的机制， TRiC行动。 分析伴侣蛋白反应过程中ATP水解 循环与蛋白质折叠和释放偶联，修饰的伴侣蛋白 在这个过程中的缺陷将被审查。 这些研究将在 与原核和真核伴侣蛋白平行， 它们以类似的方式促进蛋白质折叠。 伴侣蛋白结合蛋白的构象将通过配体和 通过监测底物蛋白的抑制剂结合特性 内源性和外源性底物荧光团的荧光变化。 的 TRiC的底物谱，底物折叠和释放的动力学 将进行分析，两个结构域的融合蛋白将作为模型系统 用于域折叠的分析。 作为实现重组细胞折叠目标的重要一步， 在体外，也将在 存在大分子拥挤剂和浓缩的细胞质 蛋白质提取物，其紧密模拟细胞环境并影响 深刻地揭示了伴侣蛋白系统的特性。 这些研究的结果预计将使人们深入了解 细胞溶质蛋白质折叠的基本过程，并可能用于 更好地了解疾病相关的异常蛋白质功能。

DESCRIPTION: Double-ring chaperonin complexes are essential mediators of cellular protein folding. Based on their ability to bind unfolded polypeptides within their ring cavities, they prevent off-pathway reactions and promote productive protein folding to the native state in an ATP-dependent manner. Partial folding appears in the chaperonin cavity before release of the substrate proteins into solution. The functional principles of the bacterial GroEL/GroES chaperonin system have been derived from in vitro reconstitution studies, the exact mechanism of its action is however unclear. Unlike the homo-oligomeric GroEL, the only recently discovered eukaryotic cytosolic chaperonin TRiC is a hetero-oligomeric complex and appears not to rely on a GroES-like cofactor. Little is known about the function and substrate specificity of this chaperonin. The goal of this proposal is to understand the molecular mechanism by which chaperonin ring complexes mediate cytosolic protein folding. The following specific aspects will be addressed with the purified components in vitro: Folding assays with immobilized chaperonin complexes and trap chaperonins will be used in experiments to determine the mechanism of GroEL/GroES and TRiC action. To analyze how ATP hydrolysis during the chaperonin reaction cycle is coupled to protein folding and release, the modified chaperonins with defects in this process will be examined. These studies will be done in parallel with the prokaryotic and eukaryotic chaperonins to learn whether they facilitate protein folding in a similar manner. The conformation of chaperonin-bound proteins will be assessed by ligand and inhibitor binding characteristics of substrate proteins by monitoring fluorescence changes of intrinsic and extrinsic substrate fluorophors. The substrate spectrum of TRiC, the kinetics of substrate folding and release will be analyzed, and two-domain fusion proteins will serve as model systems for the analysis of domain-wise folding. As a major step towards the goal of reconstituting cellular folding in vitro, the proposed biochemical assays will be performed also in the presence of macromolecular crowding agents and concentrated cytoplasmic protein extracts which closely mimic the cellular milieu and affect profoundly the properties of chaperonin systems. The results of these studies are expected to give insight into the fundamental process of cytosolic protein folding and may be useful for a better understanding of disease-related abnormal protein functions.